In various contexts, it is useful to be able to determine the fertility of a male mammal. Thus, while the artificial insemination industry is concerned with the genetic makeup of individuals resulting from artificial insemination of females, it is also importantly interested in the likelihood that fertilization will take place in any event as a consequence of the artificial insemination of the female. For example, in the bovine artificial insemination industry, bulls are evaluated on the basis of the milk production of the daughters produced from the bull. However, if the fertility of the bull is low as measured by the number of times a cow must be artificially inseminated before pregnancy occurs, the value of the semen produced by the bull and therefore the value of the bull itself is low. If fertility is sufficiently low, such bulls are disposed of.
The costs resulting from inability to reliably predict fertility may be high. Currently in the bovine artificial insemination industry it takes from five to six years to detect and weed out low-fertility bulls, throughout which period the bull must be fed, housed, and otherwise cared for at considerable expense. When a breeder examines a one-year-old, sexually mature bull, the breeder's only source of information about the bull's fertility is the pedigree information available on the animal. Testicular size and other gross physical characteristics of the animal provide little or no useful information relating to fertility.
The first determination of the bull's general desirability conventionally relates to milk production. Typically the bull is bred to cows until a selected number of daughters are produced. Typically as many as 200 daughters are produced and carefully monitored. It may take as much as a year before so many daughters are produced, so that the bull is now two years old. Two additional years must then pass before the daughters themselves become sexually mature, can be impregnated, calve, and begin to produce milk. By this time the bull is approximately four years old. The lactation of the daughters is carefully monitored, and the quality of the daughters is thus evaluated. This process may take from 10 to 12 months. By this time the bull is five years old. At that time, a first decision is made as to whether to keep the bull for breeding purposes based on the quality of the milk production of daughters produced from the bull.
If the bull is kept, it is then included in the breeder's general breeding program. Only at that time is the breeding data sufficient to begin to judge the bull's fertility, as a large population of cows are inseminated with semen from the bull. By this time, the bull is almost six years old and has been held by the breeder for approximately five years after the bull had reached sexual maturity. If the bull is found to exhibit unacceptably low fertility, the breeder has spent a large amount of money and time with a low rate of return. This expenditure could have been saved had it been possible to evaluate the fertility of the bull back at the point that the bull had become sexually mature and before all of the testing relating to the milk production of the bull's daughters had been undertaken. Typically only one out of seven bulls are kept after evaluation of the bull's progeny and fertility has been completed. If at least some of the bulls eliminated could be detected as being of low fertility early in the evaluation process, considerable money could be saved.
In other contexts, it is also useful to evaluate quickly the fertility of a breeding male. Breeding males for various types of animals are sold for use with a farmer's herd or for addition to the stud string of an artificial inseminator. Those skilled in the art are aware of no means for evaluating the fertility of animals so sold unless statistical data have been amassed on the animal's past production. As a consequence, the purchase of such animals tends to be blind speculation at least with regard to fertility. The availability of a method for determining fertility in a short period of time could thus be of value both to the seller who desires to substantiate the reasonableness of a high price for his animal and to the buyer who wants to know in advance what he is getting.
In other contexts, tests for male fertility not dependent on monitoring actual impregnations would be advantageous. Thus, human fertility clinics can evaluate the sperm count of a male but have no effective current means of evaluating the capability of that sperm to fertilize an ovum in vivo. Similarly, it would be desirable to be able to evaluate the fertility of male zoo animals and other animals in which fertility cannot be determined conveniently, economically, or in a socially practical way by attempted fertilization of large numbers of females.
It is known to those skilled in the art that mammalian spermatozoa must reside for a time in the female reproductive tract before acquiring the capacity to fertilize ova. See J. M. Bedford (1970), Sperm Capacitation and Fertilization in Mammals. Biol. Reprod., Suppl. 2, 128-158. The resulting effect upon spermatozoa is called "capacitation." Capacitation seems to require the removal of components from the spermatozoa which are epididymal or seminal plasmatic in origin. See S. Aonuma et al. (1973), Studies on Sperm Capacitation: I. The relationship between a guinea pig sperm coating antigen and a sperm capacitation phenomenom. Reprod. Fertil., 35, 425-432. After capacitation has occurred, the sperm are able to undergo an acrosome reaction. The acrosome reaction releases enzymes that digest the matrix of the cumulus cells surrounding the ovum. This digestion of the matrix permits the zona pellucida to be penetrated by spermatozoa so that the sperm may make its way toward the ovum. See D. W. Fawcett (1975), The Mammalian Spermatozoon, Dev. Biol., 44, 394-436; and R. Yanagimachi (1978), Sperm-egg Association in Mammals, Curr. Top. Dev. Biol., 12, 83-105.
It is not known precisely what components of the female reproductive tract enhance the ability of sperm to undergo capacitation and the acrosome reaction. Porcine uterine fluid was found to stimulate conversion of sperm proacrosin to acrosin necessary to the acrosin reaction. See T. J. Wincek et al. (1979), Fertilization: A Uterine Glycosaminoglycan stimulates the conversion of sperm proacrosin to acrosin, Science, 203, 553-554. In the Wincek study, the active component of the uterine fluid was destroyed by testicular hyaluronidase or chondroitin ABC lyase, suggesting that a uterine glycosaminoglycan (hereinafter "GAG") was responsible. Commercially available GAGs have also been shown to accelerate conversion of proacrosin to acrosin. See R. F. Parrish et al. (1980), Glycosaminglycan Stimulation of the in vitro Conversion of Boar Proacrosin into Acrosin, J. Androl., 1, 89-95.
Other materials are known to enhance capacitation or the acrosome reaction in spermatozoa. These include follicular fluid [See R. B. L. Gwatkin and D. F. Anderson (1969), Capacitation of Hamster Spermatozoa by Bovine Follicular Fluid, Nature, (Lond.), 224, 1111-1112.] and a chondroitin sulfate proteoglycan found in bovine follicles [H. J. Grimek and R. L. Ax (1982), Chromatographic Comparison of Chondroitin-containing Proteoglycan from Small and Large Bovine Ovarian Follicles, Biochem. Biophys. Res. Comm., 104, 1401-1406; R. W. Lenz et al. (1982), Proteoglycan from Bovine Follicular Fluid Stimulates an Acrosome Reaction in Bovine Spermatozoa, Biochem. Biophys. Res. Comm., 106, 1092-1098]. Lenz et al. in the article just cited showed that pretreatment of the proteoglycan with chondroitinase ABC prevented the effect from occurring, suggesting that the GAG side-chains may be a primary factor in the reaction. The GAGs heparin, chondroitin sulfates A, B, or C, and hyaluronic acid all promoted the occurrence of acrosome reactions in bovine sperm. The potencies were related to the degree of sulfation of the GAGs. See R. R. Handrow et al. (1982 ), Structural Comparisons Among Glycosaminoglycans to Promote an Acrosome Reaction in Bovine Spermatozoa, Biochem. Biophys. Res. Comm., 107, 1326-1332.
In vitro capacitation of rabbit sperm has also been studied. With varying degrees of success, rabbit sperm capacitation has been obtained by treatment with trypsin [P. V. Dandekar and M. Gordon (1975), Electron Microscope Evaluation of Rabbit Eggs Exposed to Spermatozoa Treated with Capacitating Agents, J. Reprod. Fertil., 44, 143-146], uterine fluid [K. T. Kirton and H. D. Hafs (1965), Sperm Capacitation by Uterine Fluid or B-amylase in vitro, Science, 150, 618-619], human or rabbit follicular fluid [A. Rosado et al. (1974), Capacitation in vitro of Rabbit Spermatozoa with Cyclic Adenosine Monophosphate and Human Follicular Fluid, Fertil. Steril., 25, 821-824; J. M. Bedford (1969), Morphological Aspects of Sperm Capacitation in Mamma1s, Adv. Biosci., 4, 35-50, respectively], and high ionic strength medium [B. G. Brackett and G. Oliphant (1975), Capacitation of Rabbit Spermatozoa in vitro, Biol. Reprod., 12, 260-274.] In vitro capacitation and acrosome reaction of mouse spermatozoa has been obtained using bovine follicular fluid. See T. Iwamatsu and M. C. Chang (1969), In vitro Fertilization of Mouse Eggs in the Presence of Bovine Follicular Fluid, Nature, 224, 919-920.
Those skilled in the art are not cognizant of a method to verify the ability of spermatozoa to undergo an acrosome reaction by light microscopic examination of the spermatozoa. Furthermore, those skilled in the art are not cognizant of any method for determining the fertility of a male mammal by direct examination of sperm therefrom.